Methods for targeted transgene-integration using custom site-specific DNA recombinases

ABSTRACT

The invention relates to biotechnology and provides novel methods for sequence-specific or sequence-directed transcription activator-like effector recombinase-mediated integration of DNA sequences of interest into host genomes. The invention also provides methods of use for novel plant transformation vectors and expression cassettes, which include novel combinations of chimeric recombinases with plant expression and transformation elements. Methods for gene-targeting, DNA sequence removal, genome modification, and molecular breeding are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/793,722, filed Mar. 15, 2013 and U.S. Provisional Application No.61/801,991, filed Mar. 15, 2013, both of which are herein incorporatedby reference in their entirety.

INCORPORATION OF SEQUENCE LISTING

The sequence listing that is contained in the file named“MONS330US.txt”, which is 3,253 bytes (measured in MS-Windows) andcreated on Mar. 13, 2014, is filed herewith by electronic submission andincorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

The invention relates to the field of biotechnology.

Description of Related Art

Site-specific recombination has tremendous potential for applicationacross a wide range of biotechnology-related fields. Zinc fingernucleases (ZFNs) are synthetic proteins, containing a DNA-binding domainand a DNA-cleavage domain, that have been successfully used to enablegenome editing. Zinc finger recombinases (ZFRs) are made by fusing arecombinase catalytic domain to the N-terminus of a zinc finger (Akopianet al., 2003). Zinc fingers (ZFs) are just one among many differentprotein folds that enable proteins to bind DNA in a sequence-specificmanner. Unfortunately, DNA targeting using zinc fingers is still limitedby the difficulty in engineering novel DNA sequence specificities andsite-specific recombination in unmodified genomes is only possible ifrecombinases can be designed to recognize endogenous target sequenceswith high specificity.

DNA-binding domains from transcription activator-like effector (TALE)proteins have a significant advantage over ZF domains as TALE proteinDNA-binding domain specificity is determined by a straight-forwardcipher allowing for the design of custom DNA-binding proteins.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for integrating anucleic acid sequence into a genomic locus including transforming a hostcell with at least one donor DNA construct. In some embodiments, thedonor DNA construct includes a first TALE recombinase (TALER) targetsequence and an exogenous DNA sequence. In particular embodiments, themethod includes transforming the host cell with at least one nucleicacid sequence encoding a TALER. In one embodiment, the TALER forms partof a tetramer and mediates recombination between the first TALER targetsequence and a second TALER target sequence located in the host cellgenome. In other embodiments, the method includes identifying atransformed host cell including the donor DNA construct integrated at aselected genomic locus in said host cell.

In certain embodiments, the donor DNA construct further includes nucleicacid sequences that cause the formation of a circular intermediatecomprising the exogenous DNA and the first TALER target sequence. In yetanother embodiment, the nucleic acid sequences that cause the formationof a circular intermediate include flanking recombinase recognitionsites. In particular embodiments, the flanking recombinase recognitionsites are selected from the group consisting of Cre, FLP, phiC31, andTALER

In some embodiments, the method further includes transforming a hostcell with a nucleic acid sequence encoding a recombinase selected fromthe group consisting of Cre, FLP, phiC31, and TALER. In certainembodiments, the recombinase mediates recombination between the flankingrecombinase recognition sites, thereby excising and circularizing anintermediate sequence including the exogenous DNA sequence and the firstTALER target sequence. In particular embodiments, the excised andcircularized intermediate sequence is integrated into the host cellgenome by TALER-mediated recombination between the first and secondTALER target sequences.

In particular embodiments, the nucleic acid sequences that cause theformation of a circular intermediate include viral sequences from adouble-stranded DNA virus or a virus with a double-stranded DNAreplication state. In one embodiment, the viral sequences includegeminivirus or caulimovirus sequences. In other embodiments, the methodfurther includes transforming a host cell with a nucleic acid sequenceencoding a replication protein from the virus. In yet other embodiments,the replication protein mediates the formation of one or moredouble-stranded DNA intermediate circles including the exogenous DNAsequence and the first TALER target sequence. In still otherembodiments, the one or more double-stranded DNA intermediate circlesare integrated into the host cell genome by TALER-mediated recombinationbetween the first and second TALER target sequences.

In one embodiment, the first and second TALER target sequences include apair of TALE binding sites flanking a recombinase core sequence. Inanother embodiment, the pair of TALE binding sites is spaced from about18 bp to about 50 bp apart.

In yet another embodiment, the donor DNA construct further includes aTALER expression construct. In one embodiment, the sequence encoding aTALER is a mRNA sequence. In some embodiments, transforming a host cellincludes a method selected from the group consisting of biolisticparticle bombardment, electroporation, and Agrobacterium-mediatedtransformation.

In certain embodiments, identifying a transformed host cell includesscreening for integration of the donor DNA construct within the secondTALER target sequence in the host cell genome. In particularembodiments, screening comprises PCR, DNA sequencing, or Southernblotting. In other embodiments, identifying a transformed host cellincludes selecting for the host cell based on the expression of aselectable marker. In another embodiment, the selectable marker confersantibiotic resistance or herbicide tolerance.

In one embodiment, the donor DNA construct is circular or linear. In yetanother embodiment, the TALER is selected from the group consisting of aspecific N-terminal transcription activator-like effector recombinaseprotein (sN-TALER), a permissive N-terminal transcription activator-likeeffector recombinase protein (pN-TALER), a specific C-terminaltranscription activator-like effector recombinase protein (sC-TALER),and a permissive C-terminal transcription activator-like effectorrecombinase protein (pC-TALER). In some embodiments, the TALER includesa small serine recombinase catalytic domain selected from the groupconsisting of Gin20H106Y, GinL7C7-EE2, GinL7C7-EE3, HinB (HinH106Y), andHinC. In certain embodiments, the method further includes regenerating aplant from said transformed host cell or a progeny therefrom. Inparticular embodiments, the plant includes the donor DNA constructintegrated at a selected genomic locus.

In another aspect, the present invention provides a method for stackingtransgenic loci including transforming a first host cell that includes afirst transgenic locus at a first TALER target sequence in the firsthost cell genome. In yet another embodiment, the method includestransforming a first host cell with at least one donor circular DNAconstruct including a second TALER target sequence and a secondtransgenic locus.

In other embodiments, the method includes transforming a first host cellwith at least one nucleic acid sequence encoding a TALER. In oneembodiment, the TALER forms part of a tetramer and mediatesrecombination between the first TALER target sequence located in thefirst host cell genome and the second TALER target sequence located onthe donor circular DNA construct. In a certain aspect, the methodincludes transforming a first host cell with at least one nucleic acidsequence encoding a selectable marker.

In particular embodiments, the method includes selecting a transformedfirst host cell expressing the selectable marker. In other embodiments,the method includes screening the selected transformed first host cellfor integration of the donor circular DNA construct to identify a hostcell of a subsequent generation that includes the first transgenic locusgenetically linked to the second transgenic locus.

In another embodiment, the selectable marker confers antibioticresistance or herbicide tolerance. In yet another embodiment, screeningcomprises PCR, DNA sequencing, or Southern blotting. In one embodiment,the steps of transforming a first host cell, selecting a transformedhost cell, and screening the selected transformed first host cell arerepeated 2 or more times with further transgenic host cells including atleast a third, fourth, and fifth transgenic locus to obtain a stack ofgenetically linked transgenic loci arranged in cis.

In a particular aspect, the present invention provides a method forcreating a transgenic marker-free cell including an integrated nucleicacid sequence at a selected genomic locus. In certain embodiments, themethod includes transforming a host cell with at least one donor DNAconstruct. In particular embodiments, the donor DNA construct includes afirst TALER target sequence and an exogenous DNA sequence. In otherembodiments, the method includes transformation of a host cell with atleast one nucleic acid sequence encoding a TALER. In one embodiment, theTALER forms part of a tetramer and mediates recombination between thefirst TALER target sequence and a second TALER target sequence locatedin the host cell genome.

In some embodiments, the method includes transformation of a host cellwith at least one nucleic acid sequence encoding a selectable marker. Incertain embodiments, the method includes selecting a transformed hostcell expressing the selectable marker. In other embodiments, the methodincludes regenerating a plant from said transformed host cell, or aprogeny therefrom, in the absence of selection for expression of theselectable marker. In yet other embodiments, the method includesscreening the regenerated plant to confirm the absence of the selectablemarker. In one embodiment, the plant includes the donor DNA constructintegrated at a selected genomic locus. In one non-limiting embodiment,the method includes selecting the regenerated plant including the donorDNA construct integrated at a selected genomic locus and not containingthe selectable marker.

In some embodiments, the nucleic acid sequence encoding the selectablemarker is a circular molecule further including a third TALER targetsequence. In certain embodiments, the TALER forms part of a tetramer andmediates recombination between the third TALER target sequence and afourth TALER target sequence located in the host cell genome at a locusthat is genetically-unlinked with the second TALER target sequencelocated in the host cell genome.

In particular embodiments, the donor DNA construct is linear and furtherincludes recombinase recognition sites selected from the groupconsisting of Cre, FLP, phiC31, and TALER. In other embodiments, the DNAconstruct includes a nucleic acid sequence encoding a selectable marker.In one embodiment, the method includes transforming a host cell with anucleic acid sequence encoding a recombinase selected from the groupconsisting of Cre, FLP, phiC31, and TALER. In another embodiment, thedonor DNA construct is circular or linear. In yet another embodiment,the recombinase mediates recombination between the flanking recombinaserecognition sites thereby excising and circularizing an intermediatesequence including the exogenous DNA sequence and the first TALER targetsequence from within the nucleic acid sequence encoding the selectablemarker. In still another embodiment, the excised and circularizedintermediate sequence is integrated into the host cell genome byTALER-mediated recombination between the first and second TALER targetsequences.

In certain embodiments, the donor DNA construct is linear and furtherincludes viral sequences from a double-stranded DNA virus or a viruswith a double-stranded DNA replication state. In particular embodiments,the DNA construct is included within the nucleic acid sequence encodinga selectable marker. In some embodiments, the viral sequences includesgeminivirus or caulimovirus sequences. In one non-limiting embodiment,the method further includes transforming a host cell with a nucleic acidsequence encoding a replication protein from the virus. In still anotherembodiment, the replication protein mediates the formation of one ormore double-stranded DNA intermediate circles including the exogenousDNA sequence and the first TALER target sequence. In other embodiment,the one or more double-stranded DNA intermediate circles are integratedinto the host cell genome by TALER-mediated recombination between thefirst and second TALER target sequences.

In some embodiments, screening comprises PCR, DNA sequencing, orSouthern blotting. In certain embodiments, the selectable marker confersantibiotic resistance or herbicide tolerance.

In an additional aspect, the present invention provides a method forgenerating genomic rearrangements between two selected genomic loci in ahost cell. In one embodiment, the method includes transforming a hostcell with at least one nucleic acid sequence encoding two sets ofincompatible TALERs. In some embodiments, each TALER set forms part of aseparate tetramer. In particular embodiments, the first TALER setmediates recombination between a first TALER target sequence at a firstgenomic locus and a second TALER target sequence at a second genomiclocus. In some embodiments, the second TALER set mediates recombinationbetween a third TALER target sequence at the first genomic locus and afourth TALER target sequence at the second genomic locus.

In other embodiments, the method includes identifying a transformed hostcell including a genomic rearrangement between two selected genomic lociin said host cell. In another embodiment, identifying a transformed hostcell includes screening genomic recombination between the first genomiclocus the second genomic locus in the host cell genome. In still anotherembodiment, the screening comprises PCR, DNA sequencing, or Southernblotting.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1: A schematic representation of TALER-mediated targetedintegration of exogenous DNA into a host cell genome usingtransformation.

FIG. 2: A schematic representation of TALER-mediated gene-targeting in apMON58401 transgenic corn line.

FIG. 3: A schematic representation of TALER-mediated gene-targetingusing Agrobacterium-mediated transformation and a recombinase-generatedintermediate circular DNA molecule.

FIG. 4: A schematic representation of Recombination-Mediated CassetteExchange (RMCE).

FIG. 5: A schematic representation of Modified Recombination-MediatedCassette Exchange (mRMCE).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods for the use of sequence-specific and/orsequence-directed recombinases for the modification of a target organismgenome by manipulating the location and frequency of geneticrecombination in a cell of the organism. For instance, the inventionprovides, in one embodiment, methods for using vectors and expressioncassettes encoding combinations of sequences encoding TALE recombinases(TALERs). Methods for causing a TALER to modify a target genome are alsoprovided, as are the genomic complements of an organism modified by theuse of such a TALER. The invention thus provides tools and methods thatallow one to insert, remove, or modify genes, loci, linkage blocks, andchromosomes within an organism.

Transcription activator-like effectors (TALEs) are DNA-binding proteinsthat recognize DNA in a modular fashion using a well describedstructural specificity thereby enabling customizable DNA targeting(Moscou and Bogdanove, 2009; Boch et al., 2009). TALE nuclease (TALEN)fusion proteins have been described that are capable of creatingsite-specific DNA double-strand breaks which can enable DNA sequencemodifications at the break site (reviewed in Bogdanove and Voytas,2011). Transcription activator-like effector recombinases (TALERs) aremade by fusing a recombinase catalytic domain to a TALE protein. Use offusion proteins containing DNA-binding domains and enzymatic domains isdescribed, for example, in U.S. Patent Application Publication Nos.2012/0222143, 2012/0214228, 2012/0192301, 2012/0178169, 2012/0178131,2012/0110685, 2011/0301073, 2011/0239315, and 2011/0145940, which areincorporated herein by reference in their entirety.

Testing Strategies for TALERs

The invention provides novel uses for sequence-specific orsequence-directed TALERs for molecular breeding by providing a genomicnucleic acid sequence to be targeted by at least one such TALER, whereinthe genomic nucleic acid sequence is native or transgenic. In addition,TALERs can be customized to catalyze recombination between one or morerecognition sequences. In certain embodiments, such a custom TALER wouldhave properties making it amenable to genetic modification such that theenzyme's recognition, binding and/or recombinase activity could bemanipulated.

One aspect of this invention is to introduce into a cell a non-naturallyoccurring sequence-specific or sequence-directed TALER to modify thecell in such a way that the cell will subsequently confer a beneficialtrait in the cell, or in an organism comprised of such cells. In onenon-limiting example, the cell is a plant cell and the trait is a traitsuch as improved yield, quality or agronomic performance. The ability togenerate such a cell, or organism derived therefrom depends onintroducing the TALER using transformation vectors and cassettesdescribed herein.

Recombinases are enzymes that catalyze DNA exchange reactions betweentarget site nucleic acid sequences (see, e.g., Nern et al., 2011; andreviewed in Garcia-Otin and Guillou, 2006; and Turan and Bode, 2011).Examples of recombinases are well known in the art and can include, forinstance, Cre recombinase (see, e.g., Nagy, 2000), Tre recombinase (see,e.g., Buchholz and Hauber, 2011), Flp recombinase (Zhu and Sadowski,1995), Hin recombinase (see, e.g., Dhar et al., 2004).

The modular nature of many proteins, recombinases included, allow forthe use of common molecular biology techniques to redesign suchproteins. Native serine recombinase catalytic domains have their owntarget DNA sequence specificity. Recognition of a recombinase-specificDNA sequence is necessary for the enzyme to properly target its intendedfunction. As such, contiguous fragments of some recombinases, forexample small serine recombinases, have been identified which encode forthe catalytic recombinase domain. However, even after the DNA-bindingdomain is replaced, such a recombinase retains some DNA-bindingcapability as required for its catalytic recombinase activity. Thus, theresulting recombination site recognized by the catalytic recombinasedomain is a composite of the core catalytic DNA target sequence of therecombinase catalytic domain and any binding sites recognized bypotential protein fusion partners.

Zinc finger recombinases (ZFRs) are fusions between zinc finger (ZF)DNA-binding domains and a hyperactive catalytic domain from a serinerecombinase. ZFs functionally replace the native DNA-binding domain ofthe serine recombinase thereby changing the target sequence that therecombinase will bind and act on. Molecular evolution techniques havebeen used to alter the recombinase domains to change or remove theirspecificity. When the recombinase domain has relaxed specificity, it isable to recombine sites with different core sequences. For a recombinasedomain with little or no specificity, the recombination activity wouldbe directed to a specific sequence exclusively by the flanking ZFs.

A variable number of imperfect amino acid repeats controls TALEDNA-binding specificity (Schornack et al., 2006). Polymorphisms atrepeat positions 12 and 13 (termed the repeat-variable di-residue, orRVD) directly determine which nucleotide is recognized. Variouscombinations of amino acid pairs located at this position correspond ina one-to-one manner (one RVD to one nucleotide) with a nucleotidetargeted for binding by a TALE DNA-binding domain containing therequisite RVD (Moscou and Bogdanove, 2009; and Boch et al., 2009). Assuch, the TALE DNA-binding domains provided herein can recognize aspecific nucleotide sequence of interest within a target DNA.

The DNA-binding domain of a TALE protein can include multipleDNA-binding repeats. Each DNA-binding repeat recognizes a single basepair within a target DNA sequence, and each DNA-binding repeat caninclude a RVD which is responsible for recognizing a single base pair ina target DNA sequence. RVD amino acid pair combinations that recognize anucleotide include: histidine-aspartic acid (HD) for recognizingcytosine (C); asparagine-glycine (NG) for recognizing thymine (T);asparagine-isoleucine (NI) for recognizing adenine (A); andasparagine-asparagine (NN) for recognizing guanine (G). Additionalspecificities for the RVD amino acids in positions 12 and 13 and thecorresponding target DNA base pair have been reported (Boch et al.,2009; and Moscou and Bogdanove, 2009).

TALERs cleave then re-ligate DNA at or near a target sequence in atarget genome that exactly matches or is closely related to a specificrecognition sequence. In one embodiment, the TALERs have a restrictednumber of recombination sites per target DNA, including, for example, aplasmid or other type of vector, or a genome. In a particularembodiment, the TALER mediates recombination at a single site in thegenome. A TALER that mediates recombination between two specificrecognition sequences, such that the recognition sequence is less likelyto occur often within a target DNA, including but not limited to agenome, may be particularly useful. In another embodiment, theTALER-mediates recombination between two recognition sequences greaterthan 14 nucleic acid bases. It is recognized that the longer therecognition sequence, the less likely it is that the TALER attemptrecombination more than once in the target genome.

In one embodiment, an effective TALER comprises at least the minimalportion of a TALE required for DNA-binding linked to a recombinasedomain. Defining the minimal DNA-binding domain can be done empiricallyby making a series of truncations to a functional TALE.

In the case of TALERs with the recombinase fused to the N-terminus ofthe TALE, any of the many possible truncations of the C-terminus thatretains robust DNA-binding activity would be acceptable and functionallyequivalent. However, at the N-terminus, the truncation position canaffect the positioning of the recombinase relative to the DNA-bindingsite. Therefore, some N-terminal truncation positions may produce TALERswith essentially equivalent DNA-binding properties but differentrecombination frequencies due to the intersection of TALER DNA-bindingand positioning of the catalytic activity of the recombinase domain.However, in cases where attachment of the recombinase to the N-terminusof a truncated TALE does not augment TALE binding, N-terminaltruncations must not be so extensive that TALE binding is impaired. Incases where attachment of the recombinase to the N-terminus of atruncated TALE does augment TALE binding, even more extensivetruncations may function. Thus, experiments looking at the minimalN-terminus for TALE binding to DNA can be used to choose a range oftruncation points to attach recombinases.

In particular embodiments, a TALER can include a non-permissiverecombinase to mediate recombination between one or more recognitionsequences. Such a specific TALER would have properties making itamenable to genetic modification such that its recognition, bindingand/or recombinase activity could be manipulated.

Molecular evolution techniques have been used to alter recombinasecatalytic domains to change or remove their specificity. When therecombinase domain has relaxed specificity, it is able to recombinesites with different DNA recognition sites. For a recombinase domainwith little or no specificity, the recombination activity is permissive,and would be directed to a specific DNA sequence with the assistance ofanother DNA-binding protein. In another embodiment of the invention, apermissive recombinase is directed to a target sequence on a nucleicacid molecule by linking the recombinase to a sequence specific TALEDNA-binding protein or molecule. As an example, a TALE DNA-bindingdomain may be used to direct a permissive recombinase to a recognitionsite (i.e., “recognition sequence”) within a target sequence (see, e.g.,U.S. Patent Application Publication No. 2012/0110685 and 2012/0178169).Other types of catalytically active recombinases that would be suitablefor use with this invention include catalytically active small serinerecombinases, large serine recombinases, or tyrosine recombinases. Incertain embodiments, these recombinases can have sequence specificityand built in DNA-binding activity. Ideally, a molecular breeder of, forexample, plants, mushrooms, or animals, would have at his or herdisposal a range of TALERs by which to induce sequence- or site-specificrecombination events at, or linked to, defined sites within nucleic acidmolecules or whole genomes.

In some embodiments, the recombinase catalytic domain can be tethered byan optional polypeptide linker of variable length to the N-terminus of aTALE protein (N-TALER). In other embodiments, the recombinase catalyticdomain can be tethered by polypeptide linker of variable length to theC-terminus of a TALE protein (C-TALER). A unique advantage of C-TALERchimeras is that these allow for a wider selection of putative TALEtargeting sequences in a host genome relative to the selection of TALEtargeting sites for N-TALER chimeras. This wider selection of putativeTALE targeting sites with C-TALER chimeras is due to the lessrestrictive orientation of TALE binding sites in a TALE targetingsequence for C-TALERs. In particular, the TALE binding site of a C-TALERmonomer can be variable lengths allowing the central sequence betweenthe two TALE binding sites to be varied. In contrast, N-TALERs have arequirement of a TALE binding site which is bounded by the firstnucleotide flanking the N-TALER central sequence which should be a T orless preferably a C or even less preferably a G.

C-TALERs and N-TALERs may be used together to allow the best TALEbinding sites to be selected. The flexibility provided by having theoption of using N-TALERs, C-TALERs or combinations of C-TALERs andN-TALERs to choose recombination sites expands the number of possibleTALER recombination sites that can be effectively used and simplifiesselection of desirable sites.

The present invention also provides for use of TALER-mediatedrecombination to genetically alter expression and/or activity of a geneor gene product of interest in a tissue- or cell-type specific manner toimprove productivity or provide another beneficial trait, wherein thenucleic acid of interest may be endogenous or transgenic in nature.Thus, in one embodiment, a TALER is engineered to mediate recombinationat specific sites in a gene of interest. Genes of interest include thosefor which altered expression level/protein activity is desired. Theserecombination events can be either in coding sequences or in regulatoryelements.

This invention provides for the introduction of a TALER into a cell.Exemplary TALERs include natural and engineered (i.e., modified)polypeptides with recombinase activity such as recombinases possessingsequence motifs and catalytic activities of the GinH107Y, GinL7C7-EE2,GinL7C7-EE3, HinB(HinH106Y), and HinC variants (see Gordley et al.,2009; Gersbach et al., 2010; and Gordley et al., 2007), as well as smallserine recombinases, large serine recombinases, and tyrosinerecombinases, naturally occurring or engineered for a given targetspecificity. Contemplated recombinases include the Cre recombinase (see,e.g., Nagy, 2000), the Tre recombinase (see, e.g., Buchholz and Hauber,2011), the Flp recombinase (Zhu and Sadowski, 1995), the Hin recombinase(see, e.g., Johnson, 2004), and those recombinases known in the art.

To be effective, the catalytically active TALER must be introduced to,or produced by, a target cell. The present invention contemplatesmultiple strategies for delivery and expression of TALERs to cells.

Transient Expression of TALERs

In some embodiments, the TALER is transiently introduced into the cell.In certain embodiments, the introduced TALER is provided in sufficientquantity to modify the cell but does not persist after a contemplatedperiod of time has passed or after one or more cell divisions. In suchembodiments, no further steps are needed to remove or segregate theTALER from the modified cell.

In another embodiment, mRNA encoding the TALER is introduced into acell. In such embodiments, the mRNA is translated to produce the TALERin sufficient quantity to modify the cell but does not persist after acontemplated period of time has passed or after one or more celldivisions. In such embodiments, no further steps are needed to remove orsegregate the TALER from the modified cell.

In one embodiment of this invention, a catalytically active TALER isprepared in vitro prior to introduction to a cell, including aprokaryotic or eukaryotic cell. The method of preparing a TALER dependson its type and properties and would be known by one of skill in theart. For example, if the TALER is a chimeric recombinase with acatalytically active small serine recombinase domain, the active form ofthe TALER can be produced via bacterial expression, in vitrotranslation, via yeast cells, in insect cells, or by other proteinproduction techniques described in the art. After expression, the TALERis isolated, refolded if needed, purified and optionally treated toremove any purification tags, such as a His-tag. Once crude, partiallypurified, or more completely purified TALERs are obtained, the TALER maybe introduced to, for example, a plant cell via electroporation, bybombardment with TALER coated particles, by chemical transfection or bysome other means of transport across a cell membrane. Methods forintroducing nucleic acids into bacterial and animal cells are similarlywell known in the art. The protein can also be delivered usingnanoparticles, which can deliver a combination of active protein andnucleic acid (Torney et al., 2007). Once a sufficient quantity of theTALER is introduced so that an effective amount of in vivo recombinaseactivity is present, the target site or sites are looped out. It is alsorecognized that one skilled in the art might create a TALER that isinactive but is activated in vivo by native processing machinery; such aTALER is also contemplated by this invention.

In another embodiment, a construct that will transiently express a TALERis created and introduced into a cell. In yet another embodiment, thevector will produce sufficient quantities of the TALER in order for thedesired target site or sites to be effectively recombined. For instance,the invention contemplates preparation of a vector that can bebombarded, electroporated, chemically transfected or transported by someother means across the plant cell membrane. Such a vector could haveseveral useful properties. For instance, in one embodiment, the vectorcan replicate in a bacterial host such that the vector can be producedand purified in sufficient quantities for a transient expression. Inanother embodiment, the vector can encode a drug resistance gene toallow selection for the vector in a host, or the vector can alsocomprise an expression cassette to provide for the expression of theTALER in an organism. In a further embodiment, the expression cassettecould contain a promoter region, a 5′ untranslated region, an optionalintron to aid expression, a multiple cloning site to allow facileintroduction of a sequence encoding a TALER, and a 3′ UTR. In someembodiments, it can be beneficial to include unique restriction sites atone or at each end of the expression cassette to allow the productionand isolation of a linear expression cassette, which can then be free ofother vector elements. The untranslated leader regions, in certainembodiments, can be plant-derived untranslated regions. Use of anintron, which can be plant-derived, is contemplated when the expressioncassette is being transformed or transfected into a monocot cell.

In other embodiments, one or more elements in the vector include a TALERtarget sequence. This facilitates recombination within the expressioncassette, enabling removal and/or insertion of elements such aspromoters and transgenes. Use of recombination to modify or deletetransgenes is described, for example, in International Publication Nos.WO2001066780A3, WO2001066780A2, U.S. Patent Application Publication Nos.2008/0178348, 2005/0060769, 2001/0056583, and U.S. Pat. Nos. 6,750,379,and 6,580,019, which are incorporated herein by reference in theirentirety.

In another approach, a transient expression vector may be introducedinto a cell using a bacterial or viral vector host. For example,Agrobacterium is one such bacterial vector that can be used to introducea transient expression vector into a host cell. When using a bacterial,viral or other vector host system, the transient expression vector iscontained within the host vector system. For example, if theAgrobacterium host system is used, the transient expression cassettewould be flanked by one or more T-DNA borders and cloned into a binaryvector. Many such vector systems have been identified in the art(reviewed in Hellens et al., 2000).

In embodiments whereby the TALER is transiently introduced in sufficientquantities to modify a cell, a method of selecting the modified cell maybe employed. In one such method, a second nucleic acid moleculecontaining a selectable marker is co-introduced with the transientTALER. In this embodiment, the co-introduced marker may be part of amolecular strategy to introduce the marker at a target site. Forexample, the co-introduced marker may be used to disrupt a target geneby inserting between recombination sites. In another embodiment, theco-introduced nucleic acid may be used to produce a visual markerprotein such that transfected cells can be cell-sorted or isolated bysome other means. In yet another embodiment, the co-introduced markermay randomly integrate or be directed via a second TALER to integrate ata site independent of the primary target site. In still yet anotherembodiment, the co-introduced molecule may be targeted to a specificlocus via recombination between recognition sites of the TALER. In theabove embodiments, the co-introduced marker may be used to identify orselect for cells that have likely been exposed to the TALER andtherefore are likely to have been modified by the TALER.

Stable Expression of TALERs

In another embodiment, a circular TALER vector is stably transformedinto a cell so as to bind a recognition sequence at or near the targetsite in the host genome with a TALE DNA-binding domain as well as arecognition sequence within the vector, and the recombinase domainrecombines the two recognition sequences thereby integrating thecircular vector into the genome. In this embodiment, the design of thetransformation vector provides flexibility for when and under whatconditions the TALER is expressed. Furthermore, the transformationvector can be designed to comprise a selectable or visible marker thatwill provide a means to isolate or efficiently select cell lines thatcontain and/or have been modified by the TALER. In a certain embodiment,a linear TALER vector is stably transformed into a cell so as to bindtwo recognition sequences within the vector with a TALE DNA-bindingdomain, wherein the recombinase domain recombines the two plasmidrecognition sequences thereby circularizing the vector, after which theTALE DNA-binding domain of the TALER binds a recognition sequence at ornear the target site in the host genome as well as the recognitionsequence within the newly formed circular TALER vector, and therecombinase domain recombines the two recognition sequences therebyintegrating the newly formed circular vector into the genome.

Cell transformation systems have been described in the art anddescriptions include a variety of transformation vectors. For example,for plant transformations, two principal methods includeAgrobacterium-mediated transformation and particle gunbombardment-mediated (i.e., biolistic) transformation. In both cases,the TALER is introduced via an expression cassette. The cassette maycontain one or more of the following elements: a promoter element thatcan be used to express the TALER; a 5′ untranslated region to enhanceexpression; an intron element to further enhance expression in certaincell types, such as monocot cells; a multiple-cloning site to provideconvenient restriction sites for inserting the TALER-encoding sequenceand other desired elements; and a 3′ untranslated region to provide forefficient termination of the expressed transcript. For particlebombardment or with protoplast transformation, the expression cassettecan be an isolated linear fragment or may be part of a larger constructthat might contain bacterial replication elements, bacterial selectablemarkers or other elements. The TALER expression cassette may bephysically linked to a marker cassette or may be mixed with a secondnucleic acid molecule encoding a marker cassette. The marker cassette iscomprised of necessary elements to express a visual or selectable markerthat allows for efficient selection of transformed cells. In the case ofAgrobacterium-mediated transformation, the expression cassette may beadjacent to or between flanking T-DNA borders and contained within abinary vector. In another embodiment, the expression cassette may beoutside of the T-DNA. The presence of the expression cassette in a cellmay be manipulated by positive or negative selection regime(s).Furthermore, a selectable marker cassette may also be within or adjacentto the same T-DNA borders or may be somewhere else within a second T-DNAon the binary vector (e.g., a 2 T-DNA system).

In another embodiment, cells that have been modified by a TALER, eithertransiently or stably, are carried forward along with unmodified cells.The cells can be sub-divided into independent clonally derived lines orcan be used to regenerate independently derived organisms. Individualplants or animals or clonal populations regenerated from such cells canbe used to generate independently derived lines. At any of these stagesa molecular assay can be employed to screen for cells, organisms orlines that have been modified. Cells, organisms or lines that have beenmodified continue to be propagated and unmodified cells, organisms orlines are discarded. In these embodiments, the presence of an activeTALER in a cell is essential to ensure the efficiency of the overallprocess.

Expression Strategies for TALERs

Promoters for transformation have been described in the art; thus theinvention provides, in certain embodiments, novel combinations ofpromoters and a sequence encoding a TALER, to allow for specificallyintroducing a recombination event into endogenous DNA (i.e., a genome).In one embodiment, a constitutive promoter is cloned 5′ to aTALER-encoding gene, in order to constitutively express the TALER intransformed cells. This may be desirable when the activity of the TALERis low or the frequency of finding and recombining the target site islow. It may also be desirable when a promoter for a specific cell type,such as the germ line, is not known for a given species of interest.

In another embodiment, an inducible promoter can be used to turn onexpression of the TALER under certain conditions. For example, a coldshock promoter cloned upstream of a TALER might be used to induce theTALER under cold temperatures. Other environmentally inducible promotershave been described and can be used in a novel combination with aTALER-encoding sequence. Another type of inducible promoter is achemically inducible promoter. Such promoters can be precisely activatedby the application of a chemical inducer. Examples of chemical induciblepromoters include the steroid inducible promoter and a quorum sensingpromoter (see, e.g., You et al., 2006; U.S. Patent ApplicationPublication No. 2005/0227285). Recently it has been shown that modifiedRNA molecules comprising a ligand specific aptamer and riboswitch can beused to chemically regulate the expression of a target gene (Tucker etal, 2005; International Publication No. WO2006073727). Such ariboregulator can be used to control the expression of a TALER-encodinggene by the addition or elimination of a chemical ligand.

In other embodiments, the promoter is a tissue specific promoter, adevelopmentally regulated promoter, or a cell cycle regulated promoter.Certain contemplated promoters include ones that only express in thegermline or reproductive cells, among others. Such developmentallyregulated promoters have the advantage of limiting the expression of theTALER to only those cells in which DNA is inherited in subsequentgenerations. Therefore, a TALER-mediated genetic modification (i.e.,genetic recombination) is limited only to cells that are involved intransmitting their genome from one generation to the next. This might beuseful if broader expression of the TALER were genotoxic or had otherunwanted effects.

Another contemplated promoter is a promoter that directs developmentallyregulated expression limited to reproductive cells just before or duringmeiosis. Such a promoter has the advantage of expressing the TALER onlyin cells that have the potential to pass on their genome to a subsequentgeneration. Examples of such promoters include the promoters of genesencoding DNA ligases, recombinases, replicases, and so on.

Tissue- and development-specific promoters are additionally useful tocontrol gamete development and essentially create haploid material (akinto haploid induction in a double haploid (DH) plant). Another aspect ofthis technology that is parallel to maternal induction systems in a DHcomprises use of a pollen expressed TALER that can recombine in one ormore sites in the male gamete genome to disable fertilization.Conveniently, the resulting seed would thus not contain a gene product.Resulting haploid cells, haploid embryos, haploid seeds, haploidseedlings, or haploid plants can be chemically treated with a doublingagent. Non-limiting examples of known doubling agents include nitrousoxide gas, anti-microtubule herbicides, anti-microtubule agents,colchicine, pronamide, and mitotic inhibitors.

Other tissue/development specific control mechanisms includemanipulating pollen delay by targeting pollen development pathwayelements or cytoplasmic male sterility elements to generate male sterileplants, which has utility for eliminating manual pollination practicesin breeding and manufacturing hybrid crops.

In another embodiment, the promoter can be part of a two componentsystem and can be activated when a second component is provided. Forexample, the promoter may require a non-native transcription factor tobind and activate. This transcription factor may be provided by crossingto a line expressing the second component. In a further elaboration, thesecond component may be regulated in an environmental, tissue ordevelopmental specific manner.

In addition to promoters, this invention provides for 5′ untranslatedregions, introns and 3′ untranslated regions that can be uniquelycombined with a TALER-encoding sequence to create novel expressioncassettes with utility for genome engineering.

Transformation Methods

Methods for transforming or transfecting a cell are well known in theart. Methods for plant transformation using Agrobacterium or DNA coatedparticles are well known in the art and are incorporated herein.Suitable methods for transformation of host cells for use with thecurrent invention are believed to include virtually any method by whichDNA can be introduced into a cell (see, e.g., Miki et al., 1993), forexample by Agrobacterium-mediated transformation (U.S. Pat. Nos.5,563,055; 5,591,616; 5,693,512; 5,824,877; 5,981,840; 6,384,301;Gelvin, 2003; and Broothaerts et al., 2005) and by acceleration of DNAcoated particles (U.S. Pat. Nos. 5,015,580; 5,550,318; 5,538,880;6,160,208; 6,399,861; and 6,403,865), etc. Through the application oftechniques such as these, the cells of virtually any species may bestably transformed.

Various methods for selecting transformed cells have been described. Forexample, one might utilize a drug resistance marker such as a neomycinphosphotransferase protein to confer resistance to kanamycin or to use5-enolpyruvyl shikimate phosphate synthase to confer tolerance toglyphosate. In another embodiment, a carotenoid synthase is used tocreate an orange pigment that can be visually identified. These threeexemplary approaches can each be used effectively to isolate a cell ormulticellular organism or tissue thereof that has been transformedand/or modified by a TALER.

When a nucleic acid sequence encoding a selectable or screenable markeris inserted into a genome at the same locus as a TALER-encoding sequenceor TALER target sequence, the marker can be used to detect the presenceor absence of the TALER or its activity. This may be useful once a cellhas been modified by the TALER, and recovery is desired of a geneticallymodified cell, or a regenerated organism from such a modified cell, thatno longer contains the TALER. In other embodiments, the marker may beintentionally designed to integrate at the recombination site, such thatit can be used to follow a modified cell independent of the TALER. Themarker can be a gene that provides a visually detectable phenotype, suchas in the seed, to allow rapid identification of seeds that carry orlack the TALER gene.

This invention provides for a means to regenerate an organism from acell with a stably integrated sequence-directed recombinase. Theregenerant can then be used to propagate additional organisms.

The invention additionally provides novel plant transformation vectorsand expression cassettes which include novel combinations of a TALERwith expression and transformation elements. The invention furtherprovides methods of obtaining a cell, a whole plant or animal, and aseed or embryo that have been specifically modified using a TALER. Thisinvention also relates to a novel cell or organism containing anon-naturally occurring sequence-specific or sequence-directed TALER.

Detection of Recombinase Activity and TALER-Mediated GenomicModification in Cells

The invention also provides molecular assays for detecting andcharacterizing cells that have been modified by a TALER. These assaysinclude but are not limited to genotyping reactions, a PCR assay, asequencing reaction or other molecular assay. Design and synthesis ofnucleic acid primers useful for such assays, for instance to assay forthe occurrence of a recombination event, are also contemplated.

Genotyping can be utilized, for instance by high throughput,non-destructive seed sampling for one or more markers, such as geneticmarkers. This sampling approach permits the rapid identification of seedcomprising preferred or selected genotypes or phenotypic characters suchthat only preferred or targeted seed is planted, saving resources ongreenhouse and/or field plots. Apparatus and methods for the highthroughput, non-destructive sampling of seeds have been described. Forexample, U.S. Patent Application Publication Nos. 2006/0048247;2006/0048248; 2006/0042527; 2006/0046244; 2006/0046264; and2007/0204366; which are incorporated herein by reference in theirentirety, disclose apparatus and systems for the automated sampling ofseeds as well as methods of sampling, testing and bulking seeds.

Use of Custom TALERs in Molecular Breeding

In some embodiments, genome knowledge is utilized for targeted geneticalteration of a genome. At least one custom TALER can be designed totarget at least one region of a genome to delete that region from thegenome. This aspect of the invention may be especially useful forgenetic alterations. The resulting organism could have a modifiedphenotype or other property depending on the gene or genes that havebeen removed. Previously characterized mutant alleles or introducedtransgenes can be targeted for TALER re-design, enabling creation ofimproved mutants or transgenic lines.

In another embodiment, a gene targeted for deletion or disruption may bea transgene that was previously introduced into the target organism orcell. This has the advantage of allowing an improved version of atransgene to be introduced or by allowing removal of a selectable markerencoding sequence. In yet another embodiment, a gene targeted fordeletion or disruption via recombination is at least one transgene thatwas introduced on the same vector or expression cassette as (an)othertransgene(s) of interest, and resides at the same locus as anothertransgene. It is understood by those skilled in the art that this typeof recombination may result in deletion or insertion of additionalsequences. Thus it may, in certain embodiments, be preferable togenerate a plurality of organisms or cells in which a deletion hasoccurred, and to screen such organisms or cells using standardtechniques to identify specific organisms or cells that have minimalalterations in their genomes following recombination. Such screens mayutilize genotypic and/or phenotypic information. In such embodiments, aspecific transgene may be removed while leaving the remainingtransgene(s) intact. This avoids having to create a new transgenic linecontaining the desired transgenes without the undesired transgene.

In another aspect, the present invention includes methods for insertinga nucleic acid of interest into a specific site of an organism's genome,wherein the nucleic acid of interest is from the genome of the organismor is heterologous with respect to the organism. This invention allowsone to select or target a particular region of the genome for nucleicacid (i.e., transgene) stacking (i.e., mega-locus). A targeted region ofthe genome may thus display linkage of at least one transgene to ahaplotype of interest associated with at least one phenotypic trait, andmay also result in the development of a linkage block to facilitatetransgene stacking and transgenic trait integration, and/or developmentof a linkage block while also allowing for conventional traitintegration. In another embodiment of this invention, a pair of sequencespecific TALERs may be used to move a sequence specifying an allelecontained on a specific locus within one linkage block contained on onechromosome to the same locus within a different linkage block on thehomologous chromosome. Progeny containing the transferred allele in thenew linkage context may exhibit one or more different traits, dependingon the transferred allele and the alleles on the new linkage block.

For instance, a TALER that is specific for, or can be directed to, arecognition sequence that is upstream of the locus containing thenon-target allele is selected. A second TALER that is specific for, orcan be directed to, a recognition sequence that is downstream of thetarget locus containing the non-target allele may also be selected. TheTALERs can be selected such that they recombine in regions where thereis no homology to the non-target locus containing the target allele.Both TALERs are cloned into expression cassettes and introduced into acell using one of the methods described above. Once introduced, theTALERs are expressed based on the properties of the promoter and otherregulatory elements found in each expression cassette that comprises aTALER-encoding sequence. The TALERs can then be expressed, and canrecombine upstream and downstream of the target locus, respectively.

The suitable distance between TALE binding sites for a particular TALERtarget sequence will vary depending on the TALER architecture used. Incertain embodiments, the distance between the TALE binding sites for aparticular TALER target sequence (binding sites begin with the 5′ T andend with the last nucleotide contacted by a TALE RVD repeat) can be 18to 50 bp. In other embodiments, the distance can be 18, 20 or 40 bp. Inyet other embodiments, the distance can be from about 18 bp to about 50bp.

In some embodiments, TALER target sequences with a preferreddi-nucleotide at or near the center of the TALER central sequencebetween the TALE binding sites can be chosen. In particular embodiments,the di-nucleotides are AT, AA, TT, TC or GA. In other embodiments, thedi-nucleotides are AT, AA and TT.

Use of TALERs in Trait Integration

Directed insertion via custom TALERs for at least one recognitionsequence in the genome, allows for targeted insertion of multiplenucleic acids of interest (i.e., a trait stack or mega-locus) to beadded to the genome of a plant or animal, in either the same site ordifferent sites. Sites for targeted integration can be selected based onknowledge of the underlying breeding value, transgene performance inthat location, underlying recombination rate in that location, existingtransgenes in that linkage block, or other factors. Once the stackedorganism is assembled, it can be used as a trait donor for crosses togermplasm being advanced in a breeding pipeline or be directly advancedin the breeding pipeline.

The present invention includes methods for inserting at least onenucleic acid of interest into at least one site, wherein the nucleicacid of interest is from the genome of an organism, such as a QTL orallele, or is transgenic in origin. A targeted region of the genome maythus display linkage of at least one transgene to a haplotype ofinterest associated with at least one phenotypic trait (as described inU.S. Patent Application Publication No. 2006/0282911), development of alinkage block to facilitate transgene stacking and transgenic traitintegration, development of a linkage block to facilitate QTL orhaplotype stacking and conventional trait integration, and so on.

In another embodiment of this invention, a pair of sequence-specificTALERs can be used to move an allele at a specific locus within onelinkage block contained on one chromosome to the same locus within adifferent linkage block on the homologous chromosome by making use ofknowledge of genomic sequence information and the ability to designcustom TALER TALE DNA-binding domains as described in the art. A TALEDNA-binding domain that is specific for, or can be directed to, arecognition sequence that is upstream of the locus containing thenon-target allele is selected from a library of TALE DNA-binding domainsor engineered as necessary. A second TALE DNA-binding that is specificfor, or can be directed to, a recognition sequence that is downstream ofthe target locus containing the non-target allele is also selected orengineered. The TALERs may be selected such that they bind in regionswhere there is no homology to the non-target locus containing the targetallele. Both TALERs may be introduced into a cell using one of themethods described above.

In another aspect, this technology enables the identification of the oneor more loci in a genome to be used for transgene insertion.Site-directed integration allows the comparison of one or moretransgenes inserted in the same position across multiple germplasm aswell as comparison of different expression elements in a transgenicconstruct. For example, 10, 100, 1000, 10,000 or 100,000 custom TALERscan be generated and used for target integration of at least oneconstruct. The recognition sequence for a TALER can be artificiallyintroduced into the genome and resulting events can be screened ormultiple custom TALE DNA-binding domains for corresponding uniquerecognition sequences can be generated.

At least one expression construct encoding at least one nucleic acid ofinterest may be evaluated for position effects to determine a preferredlocation for integration of sequences of that construct, thus allowingfor enhanced breeding efficiency, including more efficient traitintegration than the current state of the art that typically relies onrandom integration, and thus does not allow for such controlled testingand comparison. In addition, by being able to target a given insertionsite or locus of interest, variations of a given recombinant constructdesigned to insert into or otherwise manipulate genomic nucleic acidsequence at the locus of interest, and for instance comprising alternategenetic regulatory elements such as an alternate promoter or terminator,may then be tested at the given locus. The described methods thusfurther allow for the above multivariate experiments to be conductedacross germplasm, wherein position effects, promoter effects, and so onare tested in at least two different germplasm entries. Custom TALERsallow testing for the identification of identified insertion sites forthe performance of one or more transgenes. Methods and compositionsrelating to breeding for improved transgene performance are provided inU.S. Patent Application Publication No. 2009/2481438, which isincorporated herein by reference. Custom TALERs enable experiments tocompare different insertion sites as well as different construct designat the same insertion site, further facilitating development ofgermplasm-transgene combinations for enhanced transgene performance.

Further, as described herein, this process can be conductedsimultaneously or serially with manipulation of the DNArepair/recombination pathways to increase the efficiency of targetedinsertion.

The ability to execute targeted integration relies on the action of theTALE DNA-binding domain and the recombinase domain of the TALER. Thisadvantage provides methods for engineering organisms of interest,including a plant or animal or a cell, comprising at least one genomicmodification.

The present invention also contemplates that one or more geneticelements involved in DNA repair, recombination, or meiosis may bemanipulated using gene suppression, transgenic expression constructs,and/or at least one other TALER to target the genetic element. Thisstrategy can direct the outcome of the TALER-induced recombination eventto favor targeted integration or deletion. Once the action of the TALERhas occurred, the result is a non-naturally occurring modified cell.Organisms derived from and/or containing this cell can thus display atrait of interest, such as enhanced yield, quality or agronomicperformance.

In the course of using TALERs to target insertion to specific sequences,coupling targeted integration with recombination control permits therapid generation of inbreds, eliminating the need for selfing orrecurrent selection. The methods of this invention also enables traitintegration on segregating material, saving time and resources in abreeding program and enabling rapid development of sister lines. Stepsmay include, but are not limited to, the use of a positive-negativeselection system (Lida et al., 2004) or suppression of certain pathwaygenes. Methods for over-expression or suppression are known to thoseskilled in the art.

In another aspect, the present invention provides methods forcontrolling the rate of recombination in the genome of a crop plant. Inone embodiment, recombination rate for at least one genomic region ofinterest is increased in order to increase the number of potentialrecombinants at the genomic region.

In another embodiment, recombination is inhibited thus fixing the genomeof an organism in one step. In a particular embodiment, recombination isinhibited after targeted insertion of one or more nucleic acids ofinterest, as enabled by an engineered TALER (i.e., a custom TALEDNA-binding domain fused to a recombinase). This can be accomplished,for instance, by co-transformation or by achieving directedrecombination via action of a TALER, and subsequently by administrationof recombination and/or meiosis inhibition agents, such as a transgenicapproach based on manipulation of a gene involved in meiosis or DNArepair. This combination of technologies provides a strategy for“instant” trait integration.

This present invention combines tools for site-directed gene integrationas well as manipulation of recombination rate (i.e., inhibition orenhancement), for instance enabling rapid trait integration whereinrecombination is inhibited by suppression or elimination of one or moreelements of meiosis or by using approaches, such as production of adihaploid, to rapidly generate an inbred or homozygous line displaying atrait of interest. Trait integration, especially for two or more traits,is time consuming and resource intensive. The present invention advancesthe state of the art of transgenic breeding by combining methods forrecombination inhibition with methods for directed recombination, i.e.,targeted gene integration.

A custom TALER can be utilized to generate at least one trait donor tocreate a custom transgenic event that is then crossed into at least onesecond organism of interest, including a plant or animal, wherein TALERdelivery can be coupled with the nucleic acid of interest to beinserted. In other aspects one or more organisms of interest aredirectly transformed with the TALER and at least one nucleic acid ofinterest for directed insertion. It is recognized that this method maybe executed in various cell, tissue, and developmental types, includinggametes. It is further anticipated that one or more of the elementsdescribed herein may be combined with use of promoters specific toparticular cells, tissues, organs and/or development stages, such as ameiosis-specific promoter.

In certain aspects, the TALER and recombination inhibition elements aredelivered simultaneously though not necessarily expressedsimultaneously. Alternatively, the site-directed integration andrecombination inhibition elements are delivered separately. In addition,any of the steps described above may be carried out at any stage ofdevelopment, including gametes, embryos, cell culture, other tissues,and organisms. In certain aspects, cells are provided that have beenmodified to confer an improved trait. Taken together, the inventionenables a plant or animal breeder to use new tools and efficiencies formanipulating a genome within a germplasm pool.

In addition, the invention contemplates the targeting of a transgenicelement already existing within a genome for deletion or disruption.This allows, for instance, an improved version of a transgene to beintroduced, or allows selectable marker removal. In yet anotherembodiment, a gene targeted for deletion or disruption via recombinationis at least one transgene that was introduced on the same vector orexpression cassette as (an)other transgene(s) of interest, and residesat the same locus as another transgene. In one embodiment, thetransgene(s) can be deleted through the action of TALERs, as describedabove, independent of homologous recombination pathways.

In one aspect, the invention thus provides a method for modifying alocus of interest in a cell comprising (a) identifying at least onelocus of interest within a DNA sequence; (b) creating a modifiednucleotide sequence, in or proximal to the locus of interest, thatincludes a recognition sequence for a first recombinase according to theinvention; (c) introducing into at least one cell the recombinase,wherein the recombinase is expressed transiently or stably; (d) assayingthe cell for a recombinase-mediated modification in the DNA making up orflanking the locus of interest; and (e) identifying the cell or aprogeny cell thereof as comprising a modification in said locus ofinterest.

Further provided is a method for modifying a locus of interest in a cellcomprising (a) identifying at least one locus of interest within a DNAsequence; (b) creating a modified nucleotide sequence at the locus ofinterest, in or proximal to the locus of interest, that includes arecognition sequence for a first chimeric recombinase according to theinvention; (c) introducing into at least one cell the chimericrecombinase, wherein the chimeric recombinase is expressed transientlyor stably; (d) assaying the cell for a modification caused by thechimeric recombinase in the DNA sequence making up or flanking the locusof interest; and (e) identifying a cell or a progeny cell thereof ascomprising a modification in said locus of interest.

A third aspect provides a method for modifying a locus of interest in acell comprising (a) identifying at least one locus of interest within aDNA sequence; (b) identifying at least one chimeric recombinaserecognition sequence within the locus of interest; (c) introducing intoat least one cell at least one chimeric recombinase according to theinvention, wherein the cell comprises the recognition sequence in orproximal to the locus of interest and the chimeric recombinase isexpressed transiently or stably and creates modified site that includesat least one recognition sequence for the chimeric recombinase; (d)assaying the cell for a chimeric recombinase-mediated modification inthe DNA making up or flanking the locus of interest; (e) identifying acell or a progeny cell thereof which comprises a modified nucleotidesequence at said locus of interest and (f) introducing into theidentified cell at least another chimeric recombinase which recognizesthe modified nucleotide sequence at the locus of interest.

The invention further provides a method comprising one or more stepssubsequent to step (f), wherein the locus which comprises the sequencerecognized by this other chimeric recombinase is further modified. Thussequential modification of a locus of interest, by two or more chimericrecombinase according to the invention, is contemplated, and genes orother sequences added by the action of such a first chimeric recombinasemay be retained, further modified, or removed by the action of a secondchimeric recombinase. Sequences, including modified sequences, at alocus of interest may also be modified or removed, or alternativelyretained, during subsequent breeding or other crop developmentactivities, for instance with or without further use of a chimericrecombinase.

Definitions

The definitions and methods provided define the present invention andguide those of ordinary skill in the art in the practice of the presentinvention. Unless otherwise noted, terms are to be understood accordingto conventional usage by those of ordinary skill in the relevant art.Definitions of common terms in molecular biology may also be found inAlberts et al., Molecular Biology of The Cell, 5th Edition, GarlandScience Publishing, Inc.: New York, 2007; Rieger et al., Glossary ofGenetics: Classical and Molecular, 5th edition, Springer-Verlag: NewYork, 1991; King et al, A Dictionary of Genetics, 6th ed., OxfordUniversity Press: New York, 2247; and Lewin, Genes IX, Oxford UniversityPress: New York, 2007. The nomenclature for DNA bases as set forth at 37CFR § 1.822 is used.

As used herein, “domain” refers to a polypeptide that includes an aminoacid sequence of an entire polypeptide or a functional portion of apolypeptide. Certain functional subsequences are known, and if they arenot known, can be determined by truncating a known sequence anddetermining whether the truncated sequence yields a functionalpolypeptide.

As used herein, “TALE protein” refers to a transcription activator-likeeffector (TALE) protein originally identified as a virulence factor fromthe phytopathogenic bacterial genus Xanthomonas or Ralstonia. TALEproteins bind DNA in the nucleus, via a domain of DNA-binding repeats,where they act as transcriptional activators thereby contributing tovirulence.

As used herein, “TALER site” or “TALER recombination site” refers to asequence that comprises a TALER central sequence and can be recombinedby a TALER or a set of TALERs.

As used herein, “TALE DNA-binding domain” refers to the domain of a TALEprotein, or chimeric TALE-recombinase (TALER) protein, that binds to aspecific DNA sequence, defined herein as a “TALE binding site” (TBS),via a domain of DNA-binding repeats. As used herein, “DNA-bindingrepeat” refers to a sequence containing a variable number (typically 34)of amino acids, typically found in the context of animperfectly-repeating set. Each DNA-binding repeat can includehypervariable amino acid residues, defined herein as “repeat-variabledi-residues” (RVDs), typically at positions 12 and 13.

As used herein, “TALER” refers to a chimeric protein which combines atleast a first hyperactive recombinase catalytic domain from arecombinase tethered, by an optional polypeptide linker of variablelength, to the N- or C-terminus of a TALE protein.

As used herein, “recombinase core sequence” is defined as therecombination-site DNA sequence minimally required for recognition as asubstrate for a recombinase catalytic domain. As used herein, “TALERtarget sequence” refers to a nucleic acid sequence encoding a TALEbinding site followed by an optional spacer followed by a recombinasecore sequence followed a spacer sequence followed by a TALE bindingsite.

As used herein, “TALER central sequence” refers to a nucleic acidsequence flanked by TALER binding sites. In some embodiments, the TALERcentral sequence contains a recombinase core sequence with flanking,adjoining, optional spacer. In other embodiments, the TALER centralsequence of one TALER site does not contain a core sequence but can berecombined with a second TALER site that does contain a core sequence.As used herein, “TALER expression construct” refers to a DNA constructthat includes an encoded chimeric TALER protein that can be transcribed.

As used herein, “TALER reporter construct” refers to a DNA constructthat includes synthetic TALER target sequences where two TALE bindingsites, flanking a recombinase core sequence, are oriented such that therecombinase domains of the TALER proteins, when bound to the DNA, willbe positioned at the recombinase core sequence between the two TALEbinding sites (Table 1). In certain embodiments, the TALER reporterconstructs described herein include a recombinase core sequence, that isrecombined by the native Gin recombinase, and a 5′ and 3′ spacersequence (Table 1).

As used herein, “spacer” refers to a nucleotide sequence between a TALEbinding site and a recombinase core sequence (Table 1).

As used herein, “linker” refers to an amino acid sequence tethering therecombinase catalytic domain to the TALE protein.

As used herein, “perfect Gin recombinase sequence” or refers to arecombinase core sequence that is efficiently recombined with itself bya permissive or stringent, hyperactive Gin recombinase. As used herein,“native Gin recombinase sequence” refers to all or part of the sequencethat is the natural target of recombination of the Gin recombinase or avariant of that sequence where the central dinucleotide site ofrecombination is AT, AA, or TT.

As used herein, “exogenous DNA sequence” refers to DNA that is producedoutside a cell. The sequence of such DNA may be obtained from adifferent species (i.e., transgenes) or the same species (i.e., cisgenes) as the species of the cell into which it is being delivered.

A palindromic sequence is a nucleic acid sequence that is the samewhether read 5′ to 3′ on one strand or 3′ to 5′ on the complementarystrand with which it forms a double helix. A nucleotide sequence is saidto be a palindrome if it is equal to its reverse complement. Apalindromic sequence can form a hairpin. Thus, as used herein, a“pseudo-palindrome sequence” refers to an imperfect palindromic sequencewherein not all the nucleic acid base pairs obey a hairpin two-foldsymmetry.

As used herein, a “selectable marker” refers to a sequence or genecassette that facilitates the recovery of a transformed cell.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the concept, spirit andscope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

Example 1

TALER-Mediated Targeted Integration of Exogenous DNA into a SpecifiedHost Genomic Locus

DNA constructs have been designed to integrate, by TALER-mediatedrecombination, a green fluorescent protein (GFP) coding sequence (CDS)downstream from a strong constitutive promoter (e35S) driving expressionof a CP4-EPSPS CDS in a corn transgenic line. Correctly targeted eventswould result in e35S-driven GFP expression and detectable fluorescence(FIG. 2). Because TALER-mediated integration will not be directional,correctly targeted GFP integration events with improper CDS orientation,and thus no GFP expression, are expected. Transformants can be screened,by PCR or other techniques, to identify random integration eventsresulting in GFP expression.

Four target insertion sites (TR1, TR2, TR3, and TR6) have been chosen inthe transgenic corn genome; three sites (TR1, TR2, and TR3) inside theintegrated CP4-EPSPS cassette, and one site (TR6) at a separate locusoutside of the integrated CP4-EPSPS cassette. Sites TR1 and TR2 targettwo separate sites within the DnaK intron. Site TR3 targets within theCP4-EPSPS CDS. Site TR6 targets a separate corn genomic locus, the Zm5.1site. TALER-mediated recombination at TR6 will use an NptII CDS, andparomycin selection of transformants, instead of a GFP CDS. For eachtarget integration site, a TALER target sequence has been identifiedthat contains a TALER central sequence flanked by a unique pair ofN-TALER DNA-binding sites (Table 1).

TABLE 1 TALER target sequences at target integrationsites in corn genome. SEQ Tar-TALER target sequence and integration site ID get TALE bindingTALER central TALE binding NO: site site 1 sequence site 2 1 TR1TAGGGACATGGTA GATAGAACCTACAC TGCTTAGCGGTAT GTACGAAACGAA AGCAATACGAGAAATTATTTAAGCAC (Nucleotides TGTGTAATTTGG (Nucleotides  1-25 of SEQ(Nucleotides  66-90 of SEQ ID NO: 1) 26-65 of SEQ ID ID NO: 1) NO: 1) 2TR2 TCATACTACATGG GGGATTCATATTAT TACTATAATAATT GTCAATAGTATA AGGCGATGTTCGTCTGCA (Nucleotides (Nucleotides (Nucleotides 1-25 of SEQ26-45 of SEQ ID 46-70 of SEQ ID NO: 2) NO:2) ID NO: 2) 3 TR3GTGGGATGACGTT GCTTCGTCCTCTTA TGCATGCTACACG AATTGGCTCTGA AGGTCATGTCTTCTGTGCAAGCAGCC (Nucleotides GTTTCCACGGCG (Nucleotides 1-25 of SEQ (Nucleotides 66-90 of SEQ ID NO: 3) 26-65 of SEQ ID  ID NO: 3) NO: 3) 4TR6 TGGCATGATGAAG TGATGAATTCATCA TGGTACCTCTATT GCAACATGGCCA ATCAAGCTAGGTAC (Nucleotides (Nucleotides  (Nucleotides  1-25 of SEQ26-47 of SEQ ID 48-66 of SEQ ID NO: 4) NO: 4) ID NO: 4)

Previous experiments using a bacterial expression system havedemonstrated that the TALER central sequences of the TR1, TR2, TR3, andTR6 target integration sites were recombined with a perfect Gin sequenceby each of the pN-TALER chimeras tested (N-TALER-IV-1 and pN-TALER-IV-5)(see U.S. application Ser. No. 14/109,823, the contents of which arehereby incorporated by reference). In these previous experiments, whenN-TALER reporter constructs (comprising a LacZalpha gene flanked by twoN-TALER target sequences) were co-transformed with a pN-TALER expressionconstruct, successful removal (“looping out”) of the LacZalpha reportergene via recombination was confirmed by the presence of white coloniesand subsequent reporter construct sequencing. For one of the N-TALERintegration target sites, the respective TR1, TR2, or TR3 TALER centralsequences were flanked by TALE13 DNA-binding sites, and the secondN-TALER target integration site comprised a perfect Gin recombinasecentral sequence flanked by TALE13 binding sites.

For in planta TALER-mediated targeted integration, donor constructs havebeen designed to place a perfect Gin recombinase central sequencebetween the respective TR1, TR2, TR3, or TR6 TALE binding sites. Thedesign of the donor constructs targeting integration sites TR1 or TR2also includes a GPF CDS cloned downstream of the partial DnaK siteselected for targeted for integration so as to allow GFP expression inproperly integrated events (see FIG. 2). The design of the donorconstruct targeting integration site TR3 places a perfect Ginrecombinase central sequence in front of a GFP CDS lacking a startcodon. Consequently, successful recombination between the donor Ginrecombinase central sequence and the host genomic central sequence willresult in the GFP CDS integration in-frame with the start codon of theCP4-EPSPS cassette. Design of each of the donor constructs,corresponding to target integration sites TR1, TR2, TR3, and TR6,includes a KanR gene for bacterial selection and an NptII gene forparomycin resistance in planta.

In planta TALER expression cassettes can contain a functional plantpromoter (e.g., DaMV), the pN-TALER-IV-5 coding region, and a functionalplant terminator (e.g., SetI). The TALE protein that can be used is thePthXho1 TALE and the RVD repeats can be substituted with the particularrepeat array that binds the desired integration site target sequence.The TALE protein can be truncated at the same N-terminal positions aspN-TALER-IV-5 used in the bacterial TALER assays above. The TALE proteincan have a full length C-terminus. Additionally, a plant nuclearlocalization sequence (NLS) can be added to the TALER C-terminus.

TABLE 2 Examples of TALER target sequences that can beused in donor constructs with perfect Gin recombinase central sequence.SEQ Tar- TALER target sequence ID get TALE binding TALER central TALE binding NO: site site 1 sequence site 2 5 TR1 TAGGGACATGGTATCCAAAACCATGG TGCTTAGCGGTAT GTACGAAACGAA TTTACAG TTATTTAAGCAC(Nucleotides (Nucleotides (Nucleotides 1-25 of SEQ ID 26-45 of SEQ 46-70 of SEQ NO: 5) ID NO: 5) ID NO: 5) 6 TR2 TCATACTACATGGTCCAAAACCATGG TACTATAATAATT GTCAATAGTATA TTTACAG TGTTCGTCTGCA(Nucleotides (Nucleotides (Nucleotides 1-25 of SEQ ID 26-45 of SEQ 46-70 of SEQ NO: 6) ID NO: 6) ID NO: 6) 7 TR3 GTGGGATGACGTTTCCAAAACCATGG TGCATGCTACACG AATTGGCTCTGA TTTACAG GTGCAAGCAGCC(Nucleotides (Nucleotides (Nucleotides 1-25 of SEQ ID 26-45 of SEQ46-70 of SEQ NO: 7) ID NO: 7) ID NO: 7) 8 TR6 TGGCATGATGAAGTCCAAAACCATGG TGGTACCTCTATT GCAACATGGCCA TTTACAG AGGTAC (Nucleotides(Nucleotides (NucleotideS 1-25 of SEQ ID 26-45 of SEQ 46-64 of SEQNO: 8) ID NO: 8) ID NO: 8)Seed from one or more corn lines made by transformation with pMON58401(U.S. Pat. No. 7,919,321 and U.S. Patent Publication No. 2011/0126310),and homozygous for a single insertion of the transgene cassette, can beobtained. Using standard protocols, callus can be generated from thistransgenic seed. The donor DNA and TALER expression cassettes targetingthe TR1, TR2, TR3 or TR6 sites can be delivered as plasmids to thecallus using a standard biolistic method, or other plant transformationprotocols. Transformation using only a donor DNA construct can be usedas a negative control. Post-transformation calli co-bombarded with donorDNA and TALER expression constructs targeting the preselected sites,TR1, TR2, TR3, or TR6, can be examined for GFP fluorescence. Callico-bombarded with donor DNA and TALER expression constructs targetingthe TR1, TR2, TR3, and TR6 sites, and exhibiting stable sectors onselection media containing paromycin, can be recovered and placed onregeneration media. After plants are regenerated, they can be assayedfor GFP fluorescence and analyzed using known molecular techniques(e.g., PCR and emulsion PCR) to assess donor and the target sitelinkage.

Example 2

TALER-Mediated Targeted Integration of Exogenous DNA into a SpecifiedHost Genomic Locus Using Recombinase-Mediated or Virus-MediatedCircularization

Recombination between a single host genomic site and a correspondingsite in a circular donor DNA molecule will result in integration of theentire donor DNA molecule into the host genome at the site ofrecombination. By contrast, recombination between a single host genomicsite and a corresponding site in a linear donor DNA molecule will resultin a chromosomal break. As many transformation methods employ linear DNAmolecules, a method to avoid host chromosomal breakage fromTALER-mediated integration of a linear donor DNA molecule is described.

An exogenous DNA sequence of interest to be integrated into a hostgenome (FIG. 3, “cargo”), and a suitable TALER target sequence site forTALER-mediated recombination, are flanked by recombination sites (e.g.,Lox sites). The corresponding recombinase (e.g., Cre) for these flankingsites is transformed along with the linear DNA molecule, either on thesame linear donor DNA molecule or separately. Recombination between thetwo flanking recombination sites will result in excision of the TALERtarget sequence and the exogenous DNA sequence of interest as anintermediate circular DNA molecule (FIG. 3). Expression of the TALERrecombinase, corresponding to the TALER target sequence on theintermediate circular DNA, will then mediate recombination with the hostgenomic TALER target sequence at the intended integration site.

Many recombinases well known in the art can be used to form theintermediate circular DNA molecule. Non-limiting examples include Cre,Flp, phiC31, and TALERs. Recombination mediated by the Cre/Lox system iswell known in the art, efficient, and can be irreversible depending onthe choice of recombinase and recombinase recognition site. If two TALERsites with “perfect” sites in their TALER central sequences flank theDNA sequence of interest to be integrated, the same TALER or TALER paircan be used to create the intermediate circle and integrate it into thehost genome. Because the recombination sites are constrained to be neareach other by being on the same linear piece of DNA, TALER-mediatedintermediate circle formation should be a very efficient.

Alternatively, the DNA construct comprising an exogenous DNA sequence ofinterest to be integrated into a host genome, and a suitable TALERtarget sequence site for TALER-mediated recombination, can also compriseviral sequences from a double-stranded RNA or DNA virus (e.g.,caulimovirus) or a virus with a double-stranded replication intermediate(e.g., a geminivirus; see, e.g., Mor et al., 2003; Willment et al.,2007; Bruce et al., 2011). The corresponding replication protein forthese viral sequences can be transformed along with the linear DNAmolecule, either on the same linear donor DNA molecule or separately.The replication protein will mediate the formation of multipledouble-stranded DNA intermediate circles comprising the TALER targetsequence and the exogenous DNA sequence of interest. Expression of theTALER recombinase, corresponding to the TALER target sequence on theintermediate circular DNA, will then mediate recombination with the hostgenomic TALER target sequence at the intended integration site.

Many geminivirus well known in the art can be used to form theintermediate double-stranded DNA circles. Non-limiting examples includemaize streak virus (MSV), bean yellow dwarf virus (BYDV), and wheatdwarf virus (WDV).

The CP4-EPSPS gene can be included in the circular donor DNA molecule toserve as a selectable marker for selection of stable transformationevents (FIG. 3). However, many other selectable markers could be used.Because the intermediate circular donor DNA molecule is integratedwithout the other linear DNA molecules, the Cre and TALER expressioncassettes can be on the same, or separate, DNA molecules. The Cre andTALER expression cassettes can be on a single T-DNA containing theLox-flanked donor DNA molecule that will become the intermediate circle.Alternatively, the two TALER cassettes can be delivered on a separateT-DNA from the donor DNA molecule. Other possible arrangements includethe Cre cassette on a separate T-DNA, the Cre cassette and one or bothof the TALER expression cassettes on circular DNA, or any combinationthereof. Alternatively, one or more of the Cre or TALERs can bedelivered as proteins. Alternatively, one or more of the Cre or TALERscan be delivered as mRNA molecules. A preferred embodiment for planttransformation would be to use Agrobacterium-mediated transformation todeliver the targeting construct carrying the DNA sequence of interest.More preferably, one embodiment would be to deliver the Cre, or otherrecombinase and TALER molecules, as expression cassettes.

Methods similar to these have been used successfully using Cre or Flprecombinases with Agrobacterium-mediated transformation to insert DNAsequences of interest into plant genomic loci requiring prior insertionof Lox or Flp recombinase recognition sites. The use of thoserecombinases, and other specific recombinases known in the art, islimited to sites in the host genome that already possess the respectiverecombinase recognition sites; a limitation not shared by the methodsdescribed herein.

Example 3

pTALER-Mediated Targeted Integration of Exogenous DNA into a SpecifiedHost Genomic Locus Using Modified Recombinase Mediated Cassette Exchange(mRMCE)

Recombination-mediated cassette exchange (RMCE) has been previouslydescribed as a method for inserting traits into desired locations. Inshort, RMCE is the use of recombinases to cause recombination at twopairs of recombination sites such that a piece of DNA flanked by thesesites in a donor molecule replaces a segment in the target molecule thatis flanked by a corresponding pair of recombination sites (FIG. 4).There should be one recombination site from each pair on both the donorand target molecule.

RMCE requires a pair of recombination sites in the target molecule(e.g., a chromosome) flanking the desired site of integration. This is asignificant limitation because the likelihood of two recombination sitesfor known recombinases pre-existing at a desired genomic location isextremely low. As a result, for successful RMCE using knownrecombinases, a pair of recombination sites for the known recombinasesmust first be added to the genomic site of interest so as to enablesubsequent targeted insertions.

To overcome this limitation, a modified version of RMCE (mRMCE) isdescribed herein which does not require recombination sites for knownrecombinases to be present in the host genome. Instead, pTALERs(pN-TALER or pC-TALER) can be used, thereby allowing the use ofendogenous sites in a host genome to be readily used as target sites forrecombination and integration of a donor DNA molecule (FIG. 5).

pTALER-mediated recombination can employ a first set of pTALERs, with afirst permissive recombinase catalytic domain (i.e., GinL7C7-EE2), torecombine a first target site in the genome with a first target site inthe donor. Then, a second set of pTALERs, with a second permissiverecombinase catalytic domain different from the first permissiverecombinase catalytic domain, can be used to mediate recombinationbetween a second target site in the genome with a second target site inthe donor. In this case, the second permissive recombinase catalyticdomain can be selected to be incompatible with the first permissiverecombinase catalytic domain. Alternatively, both the first and secondset of pTALERs can contain the same permissive recombinase catalyticdomain.

For mRMCE, TALERs are designed for a pair of endogenous sequencesflanking the desired genomic site of integration and the correspondingrecombination sequences in the donor molecule. At each recombinationsite, the TALE binding sites are in the appropriate orientation, andsuitably spaced, for compatibility with their respective N-TALER orC-TALER. Given the ease of identifying and designing TALE binding sites,and the flexibility provided by permissive TALERs as described above,the requisite criteria of orientation and spacing of the TALE bindingsites should not be limiting.

A key difference between the mRMCE and RMCE methods is in the selectionof host genome recombination target sites. RMCE requires that therecombination target sites comprise a recombinase-specific recognitionsequence, which generally requires an initial modification of the hostgenome to create those sequences at the desired recombination locus. Bycontrast, mRMCE allows for the recombination recognition sites in thehost genome to be selected from pre-existing endogenous sequence. Theadvantage is in the ability to target any number of host genomic locifor recombination using the endogenous sequence at these sites to guideTALER and donor DNA construct design and thus facilitate efficientTALER-mediated recombination at these sites.

In cases where mRMCE uses pTALERs sets with compatible recombinasecatalytic domains (e.g., pTALERs with the GinL7C7-EE2 domain), it can bedesirable to have different di-nucleotides at the centers of the perfectsites and corresponding genomic loci; this increases the efficiency ofdirectional integration of the donor DNA sequence of interest.

The mRMCE method can also be used with TALERs containing recombinasecatalytic domains derived from differing serine recombinases, or otherrecombinases variants (e.g., gamma delta, Tn3, Sin). TALERs possessingthese variant recombinase catalytic domains would be incompatible withTALERs possessing non-identical recombinase catalytic domains (i.e.,Gin) and therefore would not form functional tetramers. As such, TALERsets designed with incompatible recombinase catalytic domains can enabledirectional integration of donor DNA molecules.

To produce targeted transformation with the mRMCE method usingrecombinase domain variants with altered specificity, first, a hostgenomic locus for targeted integration of exogenous DNA is chosen.

mRMCE with exogenous DNA first requires the identification of two pairsof TALE binding sites, in the correct orientation and spacing to enableTALER activity, flanking the target host genomic locus. Here,consideration can be given to selecting TALER target sequences withdifferent central di-nucleotides, which can improve efficiency and bepreferable if the recombinase variants to be used are compatible.Second, two recombinase variants, and their expression constructs,suitable for mediating recombination between the TALER target sequencesare identified and created. Preferably, the two recombinase variants areincompatible (e.g., one is a modified Gin domain and the other is amodified Sin domain). Third, a host cell is transformed with a donor DNAmolecule (e.g., a T-DNA) containing the two TALER target sequences,selected from the host genomic locus of interest, flanking the sequenceto be integrated into the host genome. In the same or subsequenttransformations, the TALERs are delivered into the host cell (e.g., byplacing the TALER expression cassettes on the donor DNA molecule).Optionally, a counter-selectable marker can be placed on the donor DNAmolecule outside the two TALER target sequences so as not to beintegrated during TALER-mediated recombination. Fourth, transformationevents are recovered by selection of a selectable marker contained inthe donor DNA molecule or, alternatively, on a separate co-transformedDNA molecule. One skilled in the art will know suitable methods forrecovering such events. Finally, transformants are screened for targetedintegration events using standard molecular assays (e.g., PCR, orSouthern blotting).

To produce targeted transformation with the mRMCE method using pTALERs(e.g., both pTALERs can have the GinL7C7-EE2 domains) can be followedessentially as described above with the exception of changes to thethird step. Here, following selection of a targeted host genomic locusand corresponding TALE binding sites, TALER constructs containing thecorrect TALE binding site fused to permissive recombinase catalyticdomains are created. Ideally, two incompatible recombinase domains areused (e.g., one domain is GinL7C7 and the other is a modified Tn3 domainsuch as that previously described (Proudfoot et al., 2011)). These TALERconstructs will encode either pN-TALERs or pC-TALERs.

Example 4

Creating Genetically-Linked Mega-Loci in a Host Genome UsingTALER-Mediated Targeted Integration of Exogenous DNA

One utility of using TALERs to insert transgenes, or other sequences ofinterest, into targeted locations in a host genome is the creation of amega-locus comprising the desired sequences. The mega-locus can havemany sequences of interest (hereafter called traits), inserted intounique, separate loci in the host genome, but all genetically linked.Because they are genetically linked, they can segregate together and betransmitted to the next generation together. Small genetic distances canspan large physical distances (i.e., many base pairs), therefore,multiple individual traits can be inserted at unique locations such thatthere is minimal or no effects on site-dependent gene expression. Suchmega-loci can be built up by r-retransformation of lines containinganother trait, or by independently transforming lines that do notcontain all or any other traits of interest, and subsequently combiningthe traits onto a single chromosome by genetic recombination.

In addition TALER-mediated integration of traits, mega-loci can containtraits placed by other methods, such as nuclease-mediated targeting orpositive/negative selection of homologous recombination. Additionally,the mega-loci can be on extra chromosomes (e.g., artificial chromosomesor B chromosomes).

Example 5

Creating Genetically-Linked Mega-Loci in a Host Genome Using SequentialTALER-Mediated Targeted Integration Events of Exogenous DNA inHeterologous Chromosomes, and Meiotic Recombination

Placing two independent traits at specific positions on homologouschromosomes (within a genome) for independently transformed individuals,these can be crossed, and meiotic recombination will generate achromosome with both traits. Subsequently, additional traits can beplaced at sites that are at nearby genetic positions on homologouschromosomes in separate independent transformations, and these can becombined in a stepwise fashion to create chromosomes with many differenttraits. Traits can also be removed by meiotic recombination, or replacedwith other traits put at the same or nearby sites.

In addition to meiotic recombination, TALER-mediated recombination canbe used to link traits that are placed at different physical locationsbut identical genetic positions. For example, traits placed on eitherside of a centromere, or at two sites on the same side of a centromerewithin the recombination-free zone that surrounds centromeres, can belinked by TALER-mediated recombination between the two chromosomes at alocus between the two traits, thereby creating a mega-locus.

Example 6

Creating Marker-Free Events Using TALER-Mediated Targeted Integration ofExogenous DNA into Specified Host Genomic Loci

One-Step Method.

Recovery of transgenic events often requires selection of a gene thatalleviates the effect of one or more compounds applied to cells ortissue (e.g., in the media) that is toxic or otherwise prevents growthor proper development of a non-transformed event. Such markers include,for example, herbicide tolerance genes in plants. In some cases,persistence of the selectable marker is undesirable. A utility of themethodology described herein is to produce transformed events,containing a DNA sequence of interest integrated into a specific genomiclocus, where any selectable marker used in the process has been removedfrom the genome of the selected transformed events.

Co-transformation of a cassette, encoding a selectable marker, alongwith a DNA sequence of interest can allow events that contain targetedDNA sequences of interest to be recovered by selection. Delivery of theselectable marker cassette can be by a method that does not facilitateits integration into the targeted genomic locus while simultaneouslyemploying TALERs or other methods to cause the DNA sequence of interestto be integrated at said locus. Events that survive selection will havean increased chance of containing the sequence of interest. Followingselection, transformants would be screened for events where theselectable marker cassette and the targeted, integrated DNA sequence ofinterest are unlinked. In subsequent transformant progeny, theseunlinked events can independently segregate to give rise to marker-freetransformants containing the integrated DNA sequence of interest at theintended target locus.

In one embodiment of this method, a circular DNA molecule, containingthe DNA sequence of interest and a TALER target sequence as describedabove, is co-bombarded into a cell with separate TALER expressionconstructs and a selectable marker construct that does not contain TALERtarget sequences, and is therefore not targeted for genomic integration.

When using Agrobacterium-mediated plant transformation, the selectablemarker can be delivered on a separate T-DNA from the T-DNA containingthe precursor to the intermediate circle. Alternatively, the selectablemarker can be on the same T-DNA that contains the precursor to theintermediate circle, as long as the selectable marker is not included inthe intermediate circle.

In addition, the selectable marker can be delivered as a disruptedcassette that is repaired by removal of the intermediate circle. In thisway, all events recovered, at least transiently, will have anintermediate circle, thereby increasing the likelihood of recoveringselected transformants containing a targeted integration of the DNAsequence of interest.

In another embodiment of this method, the selectable marker can betargeted for integration at a different genomic locus than the DNAsequence of interest. Targeted integration of the selectable marker canbe by the same method or by a different method (e.g., nucleasestimulated gene-targeting, TALER-mediated insertion, homology mediatedgene-targeting) than that used to target integration of the DNA sequenceof interest.

Two-Step Method.

A two-step method is contemplated to generate marker free events. Forthis method, the selectable marker (or other sequences used to assist intransformation or gene-targeting) is flanked by recombinase recognitionsites (e.g., Lox sites). First, a donor DNA construct containing aselectable marker and the DNA sequence of interest is integrated into achosen host genomic locus via TALER-mediated recombination. Second,following selection and identification of targeted events, thesetransformants can be crossed to a second transgenic plant lineexpressing a recombinase (e.g., Cre) that will recombine the two sitesflanking the selectable marker (i.e., the Lox sites) and thus removingthe intervening selectable marker sequence.

Alternatively, expression of the recombinase used to remove theselectable marker can be tissue-specific, or chemically orenvironmentally inducible. As such, following post-transformationselection, the recombinase can be activated and remove the sequenceflanked by the recombination sites. Furthermore, the recombinaseexpression cassette itself can be included in the sequence to beremoved.

Example 7

Genomic Rearrangements Using TALERs

Recombinases can cause genomic rearrangements by recombining sites atdifferent locations in the genome. However, use of recombinases fordirected genomic rearrangements is currently not practiced because thenative recombinase target sites for known recombinases are rarely foundat the desired position of genomic rearrangement. TALERs are ideal toolsfor solving this problem due to the flexibility in altering thespecificity of the TALER to direct targeted genomic rearrangement.

As demonstrated above, only 6 to 8 bp of specificity are required in theTALER central sequence between two TALE binding sites (e.g.,NNNNNNACCNNGGTNNNNNN (SEQ ID NO:9) where NN can be at a minimum AT, AA,TT, TC, or GA) for recombination to be catalyzed by pTALERs made withthe GinL7C7-EE2 recombinase catalytic domain. sTALERs using the Gindomain have at most a 12 bp sequence requirement (NNNNAAACCNNGGTTTNNNN(SEQ ID NO:10) where NN can be at a minimum AT, AA, TT, TC, or GA).Because TALE binding sites are easily designed and there is considerableflexibility for the length of the TALER central sequence for some TALERvariants, almost any time either sequence (SEQ ID NO:9 or SEQ ID NO:10)is encountered, a TALE set can be designed to recombine it with anothersuch sequence elsewhere in the genome. Additional TALER variants can bedeveloped with less or different sequence specificity, further expandingthe possible sites in the genome that can be recombined for genomicrearrangement.

Additionally, since the small serine recombinases can be easily alteredby molecular evolution (e.g., cycles of selection of functional variantsfrom pools of alterations), TALERs using small serine recombinases arean ideal platform to make new recombinases targeting specific genomicloci for rearrangement.

To create specific genomic rearrangements, first, a preferred (perfect)recombinase core sequence (e.g., NNNNNNACCNNGGTNNNNNN (SEQ ID NO:9)where NN can be at a minimum AT, AA, TT, TC, or GA for pTALERs withGinL7C7-EE2, or NNNNAAACCNNGGTTTNNNN (SEQ ID NO: 10) where NN can be ata minimum AT, AA, TT, TC, or GA for TALERs with Gin) is identified inhost genomic loci where rearrangement is desired. If intergenicrecombination between homologous loci is desired, the same TALER targetsequence can be used on both chromosomes. Second, the appropriate TALERexpression constructs, designed to bind on either side of the endogenoussequences of interest targeted for recombination, are produced. Ifnon-homologous sites are desired, then four TALERs can be required toform a functional set. Third, these TALERs are expressed, stably ortransiently, in the host organism via transformation with TALERexpression constructs, TALER-encoding mRNA, or TALER proteins. Fourth,transformants are screened for the intended genomic rearrangement usingstandard molecular techniques (e.g., PCR, Southern blotting, or othertechniques known to one of skill in the art).

Engineered genomic rearrangements can, for example, be useful for: 1.Mimicking genetic recombination (especially between sites that are verytightly linked). 2. Undoing rearrangements that differentiate genomes ofrelatives to facilitate introgression of useful genetic material. Forexample, many plant species have wild relatives with useful agronomicproperties such as disease resistance. However, when the trait isbrought into the domesticated variety, it brings along other geneticmaterial that is linked to the trait. When rearrangements such asinversions differentiate the genomic regions of the relative with theuseful trait from the domestic variety, meiotic recombination may notoccur to unlink the undesirable genetic material. Undoing thedifferentiating rearrangement would allow recombination to occur and thedesired trait to be cleanly introgressed into the domesticated variety.3. Creating regions of reduced or eliminated genetic recombination. 4.Moving native traits from one genomic locus to another to allow for thecreation of mega-loci of useful traits and introgressed into otherlines. 5. Facilitating inter-species chromosomal exchange (e.g.,recombination between a wheat chromosome and a rye chromosome to replacea portion of the wheat chromosome with the related region from the ryechromosome).

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method of integrating a nucleic acid sequenceinto a selected genomic locus comprising: a) transforming a host cellwith: i) at least one linear donor DNA construct comprising a firstTALER target sequence and an exogenous DNA sequence, wherein the donorDNA construct further comprises nucleic acid sequences that cause theformation of a circular intermediate comprising the exogenous DNAsequence and the first TALER target sequence; ii) at least one nucleicacid sequence encoding a TALER, wherein the TALER comprises a smallserine recombinase catalytic domain and forms part of a tetramer andmediates recombination between the first TALER target sequence and asecond TALER target sequence located in the host cell genome, whereinthe second TALER target sequence is endogenous to the host cell genome;and b) identifying a transformed host cell comprising the donor DNAconstruct integrated at a selected genomic locus in said host cellwherein the first and second TALER target sequences comprise a pair ofTALE binding sites flanking a recombinase core sequence, wherein thepair of TALE binding sites are spaced from about 18 bp to about 50 bpapart.
 2. The method of claim 1, wherein the nucleic acid sequences thatcause the formation of the circular intermediate comprise flankingrecombinase recognition sites.
 3. The method of claim 2, wherein theflanking recombinase recognition sites are selected from the groupconsisting of Cre, FLP, phiC31, and TALER recognition sites.
 4. Themethod of claim 3, further comprising transforming a host cell with anucleic acid sequence encoding a recombinase selected from the groupconsisting of Cre, FLP, phiC31, and TALER.
 5. The method of claim 4,wherein the recombinase mediates recombination between the flankingrecombinase recognition sites, thereby excising and circularizing anintermediate sequence comprising the exogenous DNA sequence and thefirst TALER target sequence.
 6. The method of claim 5, wherein theexcised and circularized intermediate sequence is integrated into thehost cell genome by TALER-mediated recombination between the first andsecond TALER target sequences.
 7. The method of claim 1, wherein thenucleic acid sequences that cause the formation of a circularintermediate comprise viral sequences from a double-stranded DNA virusor a virus with a double-stranded DNA replication state.
 8. The methodof claim 7, wherein the viral sequences comprise geminivirus orcaulimovirus sequences.
 9. The method of claim 7, further comprisingtransforming a host cell with a nucleic acid sequence encoding areplication protein from the virus.
 10. The method of claim 9, whereinthe replication protein mediates the formation of one or moredouble-stranded DNA intermediate circles comprising the exogenous DNAsequence and the first TALER target sequence.
 11. The method of claim10, wherein the one or more double-stranded DNA intermediate circles areintegrated into the host cell genome by TALER-mediated recombinationbetween the first and second TALER target sequences.
 12. The method ofclaim 1, wherein the donor DNA construct further comprises a TALERexpression construct.
 13. The method of claim 1, wherein the sequenceencoding a TALER is a mRNA sequence.
 14. The method of claim 1, whereintransforming a host cell comprises a method selected from the groupconsisting of biolistic particle bombardment, electroporation, andAgrobacterium-mediated transformation.
 15. The method of claim 1,wherein identifying a transformed host cell comprises screening forintegration of the donor DNA construct within the second TALER targetsequence in the host cell genome.
 16. The method of claim 15, whereinscreening comprises PCR, DNA sequencing, or Southern blotting.
 17. Themethod of claim 1, wherein identifying a transformed host cell comprisesselecting for the host cell based on the expression of a selectablemarker.
 18. The method of claim 17, wherein the selectable markerconfers antibiotic resistance or herbicide tolerance.
 19. The method ofclaim 1, wherein the TALER is selected from the group consisting of asN-TALER, pN-TALER, sC-TALER, and pC-TALER.
 20. The method of claim 1,wherein the TALER comprises a small serine recombinase catalytic domainselected from the group consisting of Gin20H106Y, GinL7C7-EE2,GinL7C7-EE3, HinB (HinH106Y), and HinC.
 21. The method of claim 1,further comprising regenerating a plant from said transformed host cellor a progeny therefrom, wherein the plant comprises the donor DNAconstruct integrated at a selected genomic locus.
 22. A method ofstacking transgenic loci comprising: a) transforming a first host cellthat comprises a first transgenic locus at a first TALER target sequencein the first host cell genome, wherein the first TALER target sequenceis endogenous to the host cell genome, with: i) at least one donorlinear DNA construct comprising a second TALER target sequence and asecond transgenic locus, wherein the donor DNA construct furthercomprises nucleic acid sequences that cause the formation of a circularintermediate comprising the exogenous DNA sequence and the first TALERtarget sequence; ii) at least one nucleic acid sequence encoding aTALER, wherein the TALER comprises a small serine recombinase catalyticdomain and forms part of a tetramer and mediates recombination betweenthe first TALER target sequence located in the first host cell genomeand the second TALER target sequence located on the donor circular DNAintermediate; and iii) at least one nucleic acid sequence encoding aselectable marker; b) selecting a transformed first host cell expressingthe selectable marker; and c) screening the selected transformed firsthost cell for integration of the donor circular DNA intermediate toidentify a host cell of a subsequent generation that comprises the firsttransgenic locus genetically linked to the second transgenic locus. 23.The method of claim 22, wherein the selectable marker confers antibioticresistance or herbicide tolerance.
 24. The method of claim 22, whereinscreening comprises PCR, DNA sequencing, or Southern blotting.
 25. Themethod of claim 22, wherein steps a) to c) are repeated 2 or more timeswith further transgenic host cells comprising at least a third, fourth,and fifth transgenic locus to obtain a stack of genetically linkedtransgenic loci arranged in cis.
 26. A method for creating a transgenicmarker-free cell comprising an integrated nucleic acid sequence at aselected genomic locus comprising: a) transforming a host cell with: i)at least one linear donor DNA construct comprising a first TALER targetsequence and an exogenous DNA sequence, wherein the donor DNA constructfurther comprises nucleic acid sequences that cause the formation of acircular intermediate comprising the exogenous DNA sequence and thefirst TALER target sequence; ii) at least one nucleic acid sequenceencoding a TALER, wherein the TALER comprises a small serine recombinasecatalytic domain and forms part of a tetramer and mediates recombinationbetween the first TALER target sequence and a second TALER targetsequence located in the host cell genome, wherein the second TALERtarget sequence is endogenous to the host cell genome; and iii) at leastone nucleic acid sequence encoding a selectable marker; b) selecting atransformed host cell expressing the selectable marker; c) regeneratinga plant from said transformed host cell, or a progeny therefrom, in theabsence of selection for expression of the selectable marker; d)screening the regenerated plant to confirm the absence of the selectablemarker, wherein the plant comprises the donor DNA construct integratedat a selected genomic locus; and e) selecting the regenerated plantcomprising the donor DNA construct integrated at a selected genomiclocus and not containing the selectable marker.
 27. The method of claim26, wherein the nucleic acid sequence encoding the selectable marker isa circular molecule further comprising a third TALER target sequence.28. The method of claim 27, wherein the TALER forms part of a tetramermediates recombination between the third TALER target sequence and afourth TALER target sequence located in the host cell genome at a locusthat is genetically-unlinked with the second TALER target sequencelocated in the host cell genome.
 29. The method of claim 26, wherein thedonor DNA construct further comprises recombinase recognition sitesselected from the group consisting of Cre, FLP, phiC31, and TALER, andwherein said DNA construct is comprised within the nucleic acid sequenceencoding a selectable marker.
 30. The method of claim 29, furthercomprising transforming a host cell with a nucleic acid sequenceencoding a recombinase selected from the group consisting of Cre, FLP,phiC31, and TALER.
 31. The method of claim 30, wherein the recombinasemediates recombination between the flanking recombinase recognitionsites thereby excising and circularizing an intermediate sequencecomprising the exogenous DNA sequence and the first TALER targetsequence from within the nucleic acid sequence encoding the selectablemarker.
 32. The method of claim 31, wherein the excised and circularizedintermediate sequence is integrated into the host cell genome byTALER-mediated recombination between the first and second TALER targetsequences.
 33. The method of claim 26, wherein the donor DNA constructfurther comprises viral sequences from a double-stranded DNA virus or avirus with a double-stranded DNA replication state, and wherein said DNAconstruct is comprised within the nucleic acid sequence encoding aselectable marker.
 34. The method of claim 33, wherein the viralsequences comprise geminivirus or caulimovirus sequences.
 35. The methodof claim 33, further comprising transforming a host cell with a nucleicacid sequence encoding a replication protein from the virus.
 36. Themethod of claim 35, wherein the replication protein mediates theformation of one or more double-stranded DNA intermediate circlescomprising the exogenous DNA sequence and the first TALER targetsequence.
 37. The method of claim 36, wherein the one or moredouble-stranded DNA intermediate circles are integrated into the hostcell genome by TALER-mediated recombination between the first and secondTALER target sequences.
 38. The method of claim 26, wherein screeningcomprises PCR, DNA sequencing, or Southern blotting.
 39. The method ofclaim 26, wherein the selectable marker confers antibiotic resistance orherbicide tolerance.
 40. The method of claim 1, wherein the at least onelinear donor DNA construct further comprises a selectable marker,wherein the selectable marker is not located between the nucleic acidsequences that cause the formation of the circular intermediate, wherebythe circular intermediate does not comprise the selectable marker. 41.The method of claim 22, wherein the at least one nucleic acid sequenceencoding a selectable marker is provided on the at least one donorlinear DNA construct, wherein the selectable marker is not locatedbetween the nucleic acid sequences that cause the formation of thecircular intermediate, whereby the circular intermediate does notcomprise the selectable marker.
 42. The method of claim 26, wherein theat least one nucleic acid sequence encoding a selectable marker isprovided on the at least one donor linear DNA construct, wherein theselectable marker is not located between the nucleic acid sequences thatcause the formation of the circular intermediate, whereby the circularintermediate does not comprise the selectable marker.